Adeno-associated virus (AAV) is a prime candidate vector for human gene therapy. The long term objective is an understanding of the structural and mechanistic bases of capsid-host interactions and viral assembly. This is needed to engineer modifications of the capsid to maximize AAV's potential to deliver therapeutic DNA to targeted cells afflicted with cancer or an inherited disorder. Mapping the footprint of the cellular receptor binding site is critical to modifying tissue tropism, while mapping of the neutralizing antigenic determinants is needed to engineer viruses that can reach target cells in patients previously exposed to AAV. Similar challenges will be faced with other viral vectors, for which this work will be a paradigm. The research will build upon our recent 3 Angstrom resolution crystallographic structure of AAV serotype 2 (AAV-2), and its continuing refinement and analysis. Interactions with the cellular receptor, heparan sulfate proteoglycan, will be characterized through crystallographic analysis of AAV-2 complexed with small heparan fragments, through cryo-electron microscopy (EM) of complexes with larger fragments (in collaboration with Ken Taylor), and through site-directed mutagenesis of the binding site. AAV-antibody interactions will be studied using panels of monoclonal antibodies (MAb) prepared by Barrie Carter and Jurgen Kleinschmidt. Functional epitopes will be mapped through the sequencing of mutants to be selected by viral propagation in the presence of antibodies. Physical epitopes for representative MAb will be mapped by cryo-EM-imaging, interpreted at molecular resolution using the known AAV-2 structure. Structural studies of at least one of the other 4 serotypes will be initiated. Comparative analysis will show the extent of conservation of receptor-binding regions, and reveal the extent of variability of regions most subject to immune surveillance. All of the proposed structural studies will complement and accelerate extensive efforts elsewhere to develop AAV-based therapies, by providing a structural rationale and set of limiting constraints for modifications that currently are being made by enlightened trial-and-error.